The overall aim of this research is to understand the role of nucleotides, microtubule associated proteins (MAPS) and posttranslational modifications in the regulation of microtubule-mediated functions in cells. There is increasing evidence that co-factors, MAPs and posttranslational modifications of tubulin and MAPs show age-dependent changes and some of these changes may be important in Alzheimer's Disease. Specific Aims include: 1. To continue to probe microtubule assembly in vitro using thiophosphate analogues of GTP and ATP and highly purified tubulin free of contaminating nucleotide diphosphate kinase (NDPK) and ATPase activities to further our understanding on the mechanism of nucleotide binding and hydrolysis in tubulin assembly. In addition, the effects of tubulin-stimulated ATPase (MAP-lC), kinesin (another ATPase that reacts with tubulin) and NDPK will be probed in the microtubule assembly system with thiophosphate analogues of nucleotides. 2. To use tubulin tyrosine ligase or insulin receptor kinase to add phosphotyrosine or to phosphorylate tyrosine at the carboxy terminal end of alpha-tubulin. To analyze the potential role of phosphorylation and dephosphorylation of this critical tyrosine in tubulin assembly. 3. To further characterize the mechanism of action of tubulin and MAP-2 regulation on the phosphotyrosyl phosphatase activity of the low-molecular weight polycation-stimulated protein phosphatase. These studies should help to elucidate the role of nucleotides, microtubule associated enzymes and phosphorylation on the cytoskeleton and increase our understanding of microtubule functions. They should also contribute to our understanding of age-dependent alterations of microtubules in brain.